IN VITRO ANTIBACTERIAL POTENITAL OF SIX TRADITIONAL PALESTINIAN PLANTS AGAINST STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI

Rola Jadallah*image, Tahreer Tahainehimage

Arab American University, Palestine.

 

 

Abstract

Aim and objective: Medicinal plants remain part of Palestinian ethnomedicine, including remedies used for infectious conditions. This work evaluated six commonly used plants for in vitro activity against two representative bacterial pathogens.

Methods: Ethanolic and water extracts of clove (Syzygium aromaticum), cinnamon (Cinnamomum verum), peppermint (Mentha piperita), parsley (Petroselinum crispum), green tea (Camellia sinensis), and silver nailroot (Paronychia argentea) were assessed against Staphylococcus aureus and Escherichia coli using an agar well-diffusion assay.

Results: Ethanolic preparations produced greater antibacterial effects than the corresponding aqueous preparations. Clove showed the strongest overall response, followed by cinnamon and peppermint, whereas green tea and parsley were less active and silver nailroot showed comparatively weak activity. The tested Gram-positive organism was generally more susceptible than the Gram-negative organism.

Conclusion: These in vitro findings identify several Palestinian medicinal plants, particularly clove, as candidates for further antibacterial investigation. Isolation of active constituents, mechanistic studies, and appropriate in vivo safety and efficacy testing are required before therapeutic use can be considered.

Keywords: Camellia sinensis, Cinnamomum verum, Mentha piperita, Paronychia argentea, Petroselinum crispum, Syzygium aromaticum.

 

INTRODUCTION

 

Plant-based remedies have long formed part of traditional health practices in Palestine and elsewhere. Their continued use has encouraged scientific evaluation of botanical preparations as possible sources of biologically active molecules, including compounds with antimicrobial properties. Antimicrobial resistance has weakened the usefulness of many established antibacterial drugs and has intensified the search for additional antimicrobial leads. Plant metabolites represent one potential source of chemically diverse compounds for this purpose. Previous research has documented antibacterial effects for numerous medicinal species, but activity varies with the plant, extraction procedure, concentration, and test microorganism. Direct experimental comparison is therefore important when evaluating locally used Palestinian plants1.

Various infectious diseases, including urinary tract infections (UTIs), can occur throughout one's lifetime. UTIs are experienced by roughly 10% of the population and, if left untreated, can lead to patient morbidity. Various microorganisms, including viruses, fungi, and bacteria, can cause UTIs. However, in 95% of cases, bacteria are the primary cause2. Many researchers from underdeveloped nations and advanced countries have reported antibiotic resistance against the bacteria responsible for causing UTIs, particularly beta-lactam antibiotics, which pose a global threat. This resistance is due to the emergence of beta-lactamases enzymes, such as extended-spectrum beta-lactamase (ESBL), metallo-beta-lactamases (MBLs), and AmpC beta-lactamase3,4. As a result, the limited selection of antibiotics has become a therapeutic challenge. Therefore, finding new antibacterial agents is critical, and medicinal plant extracts have become a topic of interest due to their known antimicrobial properties. Traditional systems of medicine, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, and synthetic drugs can all benefit from the great bioresource of medicinal plants. The global usage of medicinal plants or their products is increasing as patients self-medicate, regardless of informing their physicians. Physicians generally do not inquire about using herbal remedies when taking a patient's history. Nonetheless, while prescribing allopathic medicine, doctors in primary care must inquire with the patient about any prior usage of medicinal plants or preparation, especially if the patient presents with atypical signs and symptoms, as these may induce side effects and unpleasant responses5. On the contrary, the WHO Traditional Medicines Programme encourages countries to identify safe and effective remedies based on medicinal plants within traditional medicine and integrate them into primary health care. Given the high demand and limited supply of modern medicines in low-income countries, medicinal plants, which many people already utilize, could offer a viable solution to alleviate the challenges. However, before being integrated into national policies and procedures, the efficacy of medicinal plants should be evaluated at the local level6.

Syzygium aromaticum (clove)

S. aromaticum, or clove, is an aromatic spice with a long history of culinary and medicinal use. After this first mention, the species is referred to as S. aromaticum. Its phytochemistry is dominated by phenolic and volatile constituents, particularly eugenol, which has been associated with antioxidant and antimicrobial effects. In traditional medicine, clove has been used for many ailments, including toothache, digestive issues, respiratory problems, and infections. It also possesses anti-inflammatory, antimicrobial, anti-thrombotic, antioxidant, antimutagenic, and anti-ulcerogenic properties.

Clove contains several bioactive compounds, including eugenol, responsible for most of its medicinal properties. Eugenol has been shown to have analgesic, anti-inflammatory, and antioxidant effects. Additionally, clove has been found to possess antimicrobial properties and is effective against various bacteria, viruses, and fungi7. Clove, a spice widely used for medicinal purposes and food preservation, has anti-inflammatory, antimicrobial, antithrombotic, anti-oxidant, antimutagenic, and anti-ulcerogenic properties. Given its antibacterial potential, this study aimed to examine the antibacterial effects of clove against gram-negative uropathogens producing ESBL, MBL, and AmpC beta-lactamase8.

C. verum (cinnamon)

C. verum, also known as true cinnamon, is a traditional medicinal plant used forcenturies in Ayurvedic and Chinese medicine to treat various ailments. Cinnamon can be consumed in various forms, including as a spice in cooking, as a tea, or as a supplement. While more research is needed to understand the potential health benefits of C. verum fully, it has been used safely for centuries as a traditional medicinal plant. Here are some of the traditional medicinal uses of C. verum. Cinnamon is known to have carminative properties, which means that it can help alleviate gas and bloating. In addition, this substance has been employed as a remedy for digestive problems, including diarrhea. It possesses anti-inflammatory properties that effectively treat inflammatory conditions such as arthritis and joint pain. Cinnamon has been shown to improve insulin sensitivity and help regulate blood sugar levels. It may be helpful in the management of diabetes and also used to treat respiratory infections, such as colds and flu. It can help to alleviate coughing and congestion.

Besides its culinary applications, cinnamon is used in Ayurvedic medicine to address respiratory, digestive, and gynecological ailments. Practically all parts of the cinnamon tree, such as the bark, leaves, flowers, fruits, and roots, have medicinal or culinary purposes. Furthermore, the chemical composition of the volatile oils extracted from the tree's bark, leaves, and roots differs considerably, suggesting that their pharmacological effects may also vary9. The plant's many components contain the same array of hydrocarbons in variable amounts, including main compounds such as cinnamaldehyde (bark), eugenol (leaf), and camphor (root)10. Consequently, cinnamon provides various oils with distinct properties, each determining its usefulness in various businesses. For example, unlike the leaf and bark, the root, which contains mostly camphor, has little commercial value11. Its chemical diversity is most likely responsible for cinnamon's vast range of therapeutic advantages.

C. verum (true cinnamon) is used as a spice and in several traditional medical systems. Hereafter referred to as C. verum, its bark is particularly rich in cinnamaldehyde, while other plant parts contain different proportions of volatile constituents such as eugenol and camphor. This chemical variation can influence biological activity.

M. piperita, commonly known as mint, is a traditional medicinal plant used for centuries to treat various ailments. Mint is known to have carminative properties, which means that it can help alleviate gas and bloating. It has also been used to treat nausea, indigestion, and other digestive issues.

Mint has been used to treat respiratory infections like colds and flu, which can help alleviate coughing and congestion. It has been used to alleviate menstrual cramps and other symptoms associated with menstruation and also help alleviate stress and anxiety. It can be consumed as tea or applied topically as an essential oil. Mint can be consumed in various forms, including as tea, an ingredient in cooking, or an essential oil. While more research is needed to understand M. piperita's potential health benefits, it has been used safely for centuries as a traditional medicinal plant. However, individuals who are pregnant, nursing, or taking medication should consult with a healthcare provider before using mint as a treatment. M. piperita (peppermint), subsequently abbreviated as M. piperita, is widely consumed as an herbal infusion and flavoring and is also used traditionally for digestive and respiratory complaints. Menthol and related terpenoids are prominent constituents of peppermint essential oil and contribute to its biological properties.

Petroselinum crispum (Parsley)

P. crispum, commonly known as parsley, is a traditional medicinal plant used for centuries to treat various ailments. Parsley has been used to aid digestion and alleviate bloating, gas, and constipation. It can also help to stimulate the appetite and promote healthy digestion. It contains diuretic properties, which can help increase urine production and promote kidney health. It has been used to treat urinary tract infections, kidney stones, and other kidney-related issues. Parsley contains high levels of vitamin C and other antioxidants, which can help to support the immune system and protect against infections and diseases. Parsley has been used topically to treat skin conditions such as acne and psoriasis, and it can help to soothe and heal the skin. Parsley can be consumed in various forms, including as a tea, an ingredient in cooking, or a supplement. While more research is needed to understand the potential health benefits of P. crispum fully, it has been used safely for centuries as a traditional medicinal plant. However, individuals who are pregnant, nursing, or taking medication should consult with a healthcare provider before using parsley as a treatment.

P. crispum (parsley), subsequently P. crispum, is both a food plant and a traditional remedy. Reported ethnomedicinal uses include digestive and urinary complaints, while its tissues contain vitamins, flavonoids and other phytochemicals that may contribute to antioxidant and antimicrobial effects.

Camellia sinensis (Green Tea)

C. sinensis, also known as the tea plant, is a traditional medicinal plant used for centuries for its numerous health benefits. C. sinensis contains antioxidants called catechins, which can help to reduce the risk of heart disease. Tea's polyphenols can also help improve cholesterol levels and blood pressure.

The caffeine and L-theanine in tea can help to improve cognitive function, alertness, and mood. Regular tea consumption has been linked to a reduced risk of developing neurological disorders such as Alzheimer's and Parkinson's disease. C. sinensis can help to stimulate the digestive system and reduce inflammation in the gut. It can also help to promote healthy bowel movements and reduce the risk of digestive disorders. Tea's antioxidants can help boost the immune system and protect against infections and diseases. It can also help to reduce inflammation in the body and promote overall health. C. sinensis contains compounds such as EGCG, which have anti-cancer properties. Evidence suggests that consuming tea regularly may decrease the likelihood of developing specific types of cancer, including breast, colon, and lung cancer. C. sinensis can be consumed in various forms, including green, black, white, and oolong. While tea is generally considered safe for most people, excessive consumption may lead to adverse effects such as insomnia, anxiety, and digestive issues. Individuals who are pregnant, nursing, or taking medication should consult with a healthcare provider before consuming C. sinensis as a treatment.

Green tea's antioxidant activity is also suggested to contribute to its protection against cardiovascular disease. Indeed, human volunteers who took green tea extract orally enhanced the resistance of plasma LDL to oxidation in vivo, which may reduce the risk of artherogenesis23. Green tea extract significantly reduced blood pressure rises in spontaneously hypertensive rats, which was linked to antioxidant characteristics24. Green tea catechins have been demonstrated to suppress tumor cell growth and increase the elimination of leukemia cells, which is connected with coronary artery disease25. Green tea has a reputed function in cancer prevention. Green tea contains anti-diabetic properties. It reduced glucose levels in diabetic mice's bloodstreams without changing insulin levels26. In normal rats, long-term treatment of green tea extract enhanced insulin sensitivity27.

Paronychia argentea (Silver nail root)

C. sinensis (tea plant), hereafter C. sinensis, is the source of green, black, white and oolong teas. Tea leaves contain catechins and other polyphenols whose biological activities have been widely investigated, including antioxidant and antimicrobial effects.

P. argentea can be consumed in various forms, including as tea or as a tincture. While it is generally considered safe for most people, pregnant, nursing, or taking medication should consult with a healthcare provider before consuming P. argentea as a treatment. It is also important to note that excessive consumption may lead to adverse effects such as nausea, vomiting, and diarrhea. P. argentea decreased and avoided the formation of urinary stones28, although the active chemicals have yet to be discovered. The presence of several flavonoids, according to study is responsible for the high antioxidant activity of Paronychia extracts29. Nevertheless, we could not locate any research on the cardiovascular characteristics of P. argentea extracts. The antioxidant, anti-enzymatic, and antibacterial bioactivities of essential oil from Thymus mastichina30. Thymus mastichina essential oil was found to be a major scavenger of nitric oxide (NO) radicals31. The other species mentioned above merit a European Union herbal monograph (formerly the Community herbal). These monographs offer the scientific judgment of the Committee on Herbal Medicinal Products on the safety and effectiveness data of a herbal ingredient and its preparations intended for medical use32.

This study aimed to evaluate the in vitro antibacterial activity of six plants commonly used in Palestinian traditional medicine against Staphylococcus aureus and Escherichia coli, two bacterial pathogens that cause various infections, including urinary tract infections, skin infections, and sepsis. We investigate the antibacterial activity of six plants commonly used in Palestinian traditional medicine against S. aureus and E. coli. The results of this study will provide valuable information on the potential use of these plants as sources of new antimicrobial agents to combat bacterial infections.

P. argentea (silver nailroot or silver nailwort), subsequently P. argentea, occurs in North Africa and the Middle East and has a history of regional medicinal use. Published work has reported flavonoids and antioxidant activity, although its antibacterial evidence base is less extensive than that of several other plants examined here.

The six plants tested in this study were clove (S. aromaticum), cinnamon (C. verum), peppermint (M. piperita), parsley (P. crispum), green tea (C. sinensis), and silver nailroot (P. argentea). These plants have been used traditionally in Palestine to treat different ailments, including bacterial infections. However, their antibacterial properties have yet to be fully explored, and there is a need to investigate their potential as sources of new antimicrobial agents.

In vitro assays using nutrient agar plates are commonly used to evaluate the antibacterial activity of plant extracts. The agar well diffusion method is one of the most commonly used methods to determine the antimicrobial activity of plant extracts. This method is based on the diffusion of the test compound from the wells into the agar medium, resulting in a zone of inhibition around the well, indicating the compound's antimicrobial activity.

 

MATERIALS AND METHODS

 

Plant materials and extraction

The six plants selected for this study were purchased from local markets in Palestine, and the plant materials were identified and authenticated by a botanist. The objective was to compare the in vitro antibacterial effects of six plants used in Palestinian traditional medicine against S. aureus and E. coli and to identify extracts that warrant further investigation as possible sources of antibacterial compounds.

Microorganisms and culture media

This study utilized two bacterial strains, S. aureus and E. coli, which were acquired from the American Type Culture Collection (ATCC). Nutrient agar (NA) was used as the medium for bacterial growth. The bacterial strains were preserved on nutrient agar slants and kept at a temperature of 4°C. Agar diffusion provides a screening approach in which antimicrobial constituents migrate through solid medium and growth inhibition is measured around the application site. In this study, inhibition-zone measurements were used alongside minimum inhibitory concentration data to compare the plant extracts. The agar well diffusion method was used to assess the antibacterial activity of the plant extracts. To prepare the nutrient agar plates, 20 mL of molten nutrient agar was poured into sterile Petri dishes and left to solidify. A bacterial suspension was made by introducing a single colony of each bacterial strain into 5 mL of sterile nutrient broth and incubating it at 37°C for 18 hours with continuous shaking.

Plant material for the six species was obtained from local Palestinian markets and authenticated by a botanist before extraction. Plant samples were shade-dried at room temperature and milled. Powdered material was extracted for 8 h in a Soxhlet apparatus using either 70% ethanol or distilled water. After filtration through Whatman No. 1 paper, solvent was removed under reduced pressure by rotary evaporation, and the dried extracts were kept at 4°C until testing.

To analyze the data obtained from the agar well diffusion assay, a one-way analysis of variance (ANOVA) was used, followed by Tukey's post hoc test. The mean values were expressed as mean±standard deviation (SD) of three independent experiments, and P values less than 0.05 were considered statistically significant.

The test organisms were S. aureus and E. coli obtained from the American Type Culture Collection (ATCC). Nutrient agar was used for cultivation, and stock cultures were maintained on nutrient-agar slants at 4°C.

For antibacterial screening, a colony of each test organism was transferred to 5 mL sterile nutrient broth and incubated at 37°C for 18 h with shaking before plate inoculation. Sterile nutrient agar (20 mL) was dispensed into Petri dishes and allowed to set. Six-millimeter wells were prepared with a sterile cork borer and loaded with 100 µL extract at 25, 50, or 100 mg/mL in DMSO. DMSO served as the negative control and ciprofloxacin (5 µg/disc) as the positive control. Plates were incubated at 37°C for 24 h, after which inhibition-zone diameters were recorded in millimeters.  

 

RESULTS AND DISCUSSION

 

The results of the agar well diffusion assay are presented in Table 1. The negative control (DMSO) did not inhibit the growth of S. aureus and E. coli. The positive control (ciprofloxacin) showed significant inhibition of bacterial growth with an average inhibition zone diameter of 22.3±0.8 mm. All six plant extracts showed inhibition of bacterial growth in a dose-dependent manner. Clove extract exhibited the highest inhibition zone diameter of 20.1±1.0 mm at a concentration of 100 mg/mL against S. aureus, followed by cinnamon (18.6±0.7 mm), peppermint (15.2±0.5 mm), green tea (13.5±0.4 mm), silver nailroot (9.8±0.3 mm), and parsley (11.7±0.6 mm). Against E. coli, clove also exhibited the highest inhibition zone diameter of 15.3±0.7 mm at a concentration of 100 mg/mL, followed by cinnamon (13.7±0.5 mm), peppermint (11.5±0.4 mm), green tea (10.2±0.3 mm), silver nailroot (8.6±0.2 mm), and parsley (7.8±0.3 mm).

Data from three independent experiments are reported as mean±standard deviation. Group differences were evaluated by one-way ANOVA followed by Tukey’s post hoc comparison, with p<0.05 used as the threshold for statistical significance.

Minimum Inhibitory Concentration

The results of the minimum inhibitory concentration (MIC) are shown in Table 2. The MIC values ranged from 12.5 to 100 mg/mL. Clove extract had the lowest MIC value of 12.5 mg/mL against both S. aureus and E. coli, followed by cinnamon (25 mg/mL), peppermint (50 mg/mL), green tea (50 mg/mL), silver nailroot (50 mg/mL), and parsley (100 mg/mL).

Time-Kill Assay

The results of the time-kill assay are shown in Figure 8. Clove extract at a concentration of 100 mg/mL showed the complete killing of S. aureus within 2 hours of exposure, while cinnamon and peppermint extracts showed complete killing within 4 hours. Green tea, silver nailroot, and parsley extracts showed complete killing within 8 hours of exposure. All six extracts inhibited bacterial growth, and the magnitude of inhibition increased with concentration. Clove produced the strongest overall response, followed by cinnamon and peppermint. The lower MIC observed for clove supports its comparatively greater anti-bacterial potency in this assay.

The present study investigated the antibacterial activity of six plants commonly used in traditional Palestinian medicine to treat bacterial infections. The agar well diffusion assay results demonstrated that all six plants extracts exhibited significant inhibition of bacterial growth in a dose-dependent manner. Among the plant extracts tested, clove extract was the most effective against S. aureus and E. coli, followed by cinnamon, peppermint, green tea, silver nailroot, and parsley.

The MIC values of the plant extracts ranged from 12.5 to 100 mg/mL. Clove extract had the lowest MIC value of 12.5 mg/mL against both S. aureus and E. coli, indicating its high potency against these bacteria. The time-kill assay results showed that clove extract at a concentration of 100 mg/mL showed the complete killing of S. aureus within 2 hours of exposure, suggesting its rapid bactericidal effect.

E. coli isolates from the Enterobacteriaceae family are considered one of the most significant bacterial pathogens linked with gastrointestinal tract infections and extraintestinal diseases such as urinary, respiratory, and reproductive tract infections33. Because anti-microbial agent families have a limited lifespan, it is critical to discover new ones. As a result, researchers typically concentrate on natural products to produce better treatments against multidrug-resistant bacteria strains34. Secondary metabolites of plants are examples of natural compounds employed in the pharmaceutical business35. Much research has focused on the mechanism of action of natural plant components against various bacteria to investigate their potential antimicrobial benefits36. These methods differ widely depending on the essential oils found in plants and other parts of the plant37. The antibacterial properties of plants are believed to be due to various components such as tannins, saponins, phenolic compounds, essential oils, and flavonoids, as noted in previous studies38. In this study, the primary objective was to investigate the susceptibility of a multidrug-resistant E. coli strain to different types of extracts obtained from specific medicinal plants that are abundant in Palestine. However, all aqueous extracts from the tested plant species showed no antibacterial activity, the effect against the tested E. coli strain, with similar results observed for its ethanolic extract but not its methanol extract. These findings are consistent with previous studies on these types of extractions39-41. Hence, plant extracts include various components with varying bioactivity depending on the technique of extraction used for a particular plant species. Its effectiveness may be related to the degree of solubility of certain compounds42. Moreover, the observed variance in this and other research might be connected to the chemotype, location, collection time, and vegetation cycle of the plant species under consideration43.

This investigation was limited to in vitro testing of two bacterial species and therefore cannot establish clinical efficacy, safety, pharmacokinetics, or activity against a broader pathogen panel. Future work should chemically characterize the active fractions, confirm activity using standardized susceptibility methods, investigate mechanisms of action, and evaluate safety and efficacy in appropriate in vivo models.

Limitations of the study

The study was conducted using in vitro experiments, which may not fully reflect the complexity of the human body and the potential interactions between the active compounds in the plant extracts and the host microbiota. Therefore, further studies are needed to investigate the efficacy and safety of these plant extracts in vivo. 

 

ACKNOWLEDGEMENTS

 

Authors gratefully acknowledge the technical and institutional support that facilitated this study.

 

AUTHOR'S CONTRIBUTION

 

Jadallah R: conceptualization, methodology, investigation, formal analysis, data curation, writing original draft. Tahaineh T: writing original draft, review, editing. Final manuscript was checked and approved by both authors.    

 

DATA AVAILABILITY

 

The related author can provide the empirical data supporting the study's conclusions upon request.

 

 

CONFLICT OF INTEREST

 

There are no conflicts of interest in regard to this project.

 

REFERENCES

 

  1. Moroh JLA, Fleury Y, Tia H, et al. Diversity and antibiotic resistance of uropathogenic bacteria from Abidjan. Afr J Urol 2014; 20: 18-24. https://doi.org/10.1016/j.afju.2013.11.005
  2. Farajnia S, Alikhani MY, Ghotaslou R, et al. Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran. Int J Infect Dis 2009;13: 140-4.  https://doi.org/10.1016/j.ijid.2008.04.014
  1. Baral P, Neupane S, Marasini BP, et al. High prevalence of multidrug resistance in bacterial uropathogens from Kathmandu, Nepal. BMC Res Notes 2012; 5: 38. https://doi.org/10.1186/1756-0500-5-38
  1. Sultan A, Rizvi M, Khan F, et al. Increasing antimicrobial resistance among uropathogens: Is fosfomycin the answer? Urol Ann 2015; 7: 26-30. https://doi.org/10.4103/0974-7796.148585
  1. Ahmad I, Beg AZ. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi drug resistant human pathogens. J Ethnopharmacol 2001; 74: 113-23.  https://doi.org/10.1016/S0378-8741(00)00335-4
  1. Okmen G, Mammadhkanli M, Vurkun M. The antibacterial activities of Syzygium aromaticum (L.) Merr. and Perry against oral bacteria and its antioxidant and antimutagenic activities. Int J Pharm Sci Res. 2018; 9: 4634-41.
  2. Al Omaim DA, Al Arfaj G, Abbas MAF. Knowledge and attitude of primary care physicians toward use of herbs as medicine in Al Wazarat Health Center, Prince Sultan Military Medical City, Riyadh, Saudi Arabia. Int J Pharm Sci Res. 2018; 9: 2859-68.
  3. Lazarou R, Heinrich M. Herbal medicine: Who cares? The changing views on medicinal plants and their roles in British lifestyle. Phytother Res 2019; 33: 2409-20.  https://doi.org/10.1002/ptr.6431
  1. Shen Q, Chen F, Luo J. Comparison studies on chemical constituents of essential oil from Ramulus cinnamomi and Cortex cinnamomi by GC-MS. Zhong Yao Cai. 2002; 25: 257-8.
  2. Gruenwald J, Freder J, Armbruester N. Cinnamon and health. Crit Rev Food Sci Nutr 2010; 50: 822-834. https://doi.org/10.1080/10408390902773052
  1. Paranagama PA, Wimalasena S, Jayatilake GS, et al. A comparison of essential oil constituents of bark, leaf root and fruit of cinnamon (Cinnamomum zeylanicum Blum), grown in Sri Lanka. J Natl Sci Found Sri Lanka 2010; 29: 147-153. https://doi.org/10.4038/jnsfsr.v29i3-4.2613
  1. Ferreira P, Cardoso T, Ferreira F, et al. Mentha piperita piperita essential oil induces apoptosis in yeast associated with both cytosolic and mitochondrial ROS-mediated damage. FEMS Yeast Res 2014 Nov; 14(7): 1006-1014. https://doi.org/10.1111/1567-1364.12189
  1. Rodriguez FL, Reyes EJ, Burchiel SW, et al. Risks and benefits of commonly used herbal medicines in Mexico. Toxicol Appl Pharmacol 2008 Feb 15; 227(1): 125-135.  https://doi.org/10.1016/j.taap.2007.10.005
  1. Blu Mentha PM, Goldberg A, Brinckman J. Expanded Commission E Monographs. 2nd Newton, MA: Integrative Medicine Communications; 2000.
  2. Pharmacopoeia Jugoslavica II. Pharmacopoeia of the FNRJ, Pharmacopoea jugoslavica, second edition. Belgrade: Medicinska knjiga 1951.
  3. Wichtl M, Bisset NG, et al. Herbal Drugs and Phytopharmaceuticals. Stuttgart: Medpharm Scientific Publishers; 1994.
  4. Behtash N, Kargarzadeh F, Shafaroudi H. Analgesic effects of seed extract from Petroselinum crispum (Tagetes minuta) in animal models. Toxicol Lett 2008; 180(Suppl 5): S127-S128. https://doi.org/10.1016/j.toxlet.2008.06.743
  1. Moazedi AA, Mirzaie DN, Seyyednejad SM, et al. Spasmolytic effect of Petroselinum crispum (Parsley) on rat's ileum at different calcium chloride concentrations. Pak J Biol Sci 2007; 10(22): 4036-4042.  https://doi.org/10.3923/pjbs.2007.4036.4042
  1. Aghili MH, Makhzan-al-Advia RR, Shams AMR. et al. Tehran: Tehran University of Medical Sciences; 2009: 329-330.
  2. Tonkaboni MM, Tohfeh-al-Momenin RRS, Ardekani MR, et al. Tehran: Shahid Beheshti University of Medical Sciences; 2007: 129.
  3. The cannon of medicine. Translated from Arabic to Persian by Abdulrahman Sharaf-kandi. Tehran: So-rush Publication; 1983;141.
  4. Rehecho S, Uriate-Pueyo I, Calvo J, et al. Ethnopharmacological survey of medicinal plants in Nor-Yauyos, a part of the Landscape Reserve Nor-Yauyos-Cochas, Peru. J Ethnopharmacol 2011; 133(1):75-85. https://doi.org/10.1016/j.jep.2010.09.006
  1. Miura Y, Chiba T, Miura S, et al. Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans. J Nutr Biochem. 2000; 11(4):216-22. https://doi.org/10.1016/S0955-2863(00)00068-1
  1. Negishi H, Xu JW, Ikeda K, et al. Black and green tea polyphenols attenuate blood pressure increases in stroke-prone spontaneously hypertensive rats. J Nutr 2004; 134(1):38-42.   https://doi.org/10.1093/jn/134.1.38
  1. Smith DM, Dou QP. Green tea polyphenol epigallocatechin inhibits DNA replication and consequently induces leukemia cell apoptosis. Int J Mol Med 2001; 27(6):645-52. https://doi.org/10.3892/ijmm.7.6.645
  1. Tsuneki H, Ishizuka M, Terasaw M, et al. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004; 4: 18. https://doi.org/10.1186/1471-2210-4-18
  1. Wu LY, Juan CC, Ho LT, et al. Effect of green tea supplementation on insulin sensitivity in Sprague-Dawley rats. J Agric Food Chem 2004a; 52(3):643-8. https://doi.org/10.1021/jf030365d
  1. Bouanani S, Henchiri C, Migianu GE, et al. Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. J Ethnopharmacol 2010; 129(1): 38-45.   https://doi.org/10.1016/j.jep.2010.01.056
  1. Sait S, Hamri ZS, Boulekbache ML, et al. HPLC-UV/DAD and ESI-MSn analysis of flavonoids and antioxidant activity of an Algerian medicinal plant: Paronychia argentea J Pharm Biomed Anal 2015; 111: 231-40.  https://doi.org/10.1016/j.jpba.2015.03.027
  1. Cutillas AB, Carrasco A, Martinez GR, et al. Essential oils from Murcia (Spain): Composition and antioxidant, antienzymatic and antimicrobial bioactivities. PLoS One 2018; 13(1): e0190790.  https://doi.org/10.1371/journal.pone.0190790
  1. Azazza S, El-Guendouz S, Miguel MG, et al. Antioxidant, anti-inflammatory and anti-hyperglycaemic activities of essential oils from Thymbra capitata, Thymus albicans, Thymus caespititius, Thymus carnosus, Thymus lotocephalus, and Thymus mastichina from Portugal. Natural product communications 2016; 11(7): 1934578X1601100739.  https://doi.org/10.1177/1934578X1601100739
  1. European Union monographs and list entries. 2019
  2. Beutin L. Escherichia coli as a pathogen in dogs and cats. Veterinary Res 1999; 30(2/3), 285-298.
  3. Miyasaki Y, Nichols WS, Morgan MA, et al. Screening of herbal extracts against multi-drug resistant Acinetobacter baumannii. Phytotherapy Res 2010; 24(8), 1202-1206. https://doi.org/10.1002/ptr.3113
  1. Nasim SA, Dhir B. Heavy metals alter the potency of medicinal plants. In D. Whitacre (Ed.), Reviews Environmental Contamination Toxicol 2010; 139-149).   https://doi.org/10.1007/978-1-4419-1352-4_5
  1. Montanari RM, Barbosa LC, Demuner AJ, et al. Exposure to Anacardiaceae volatile oils and their constituents induces lipid peroxidation within food-borne bacteria cells. Molecules 2012; 17(8): 9728-9740. https://doi.org/10.3390/molecules17089728
  1. Reichling J, Schnitzler P, Suschke U, et al. Essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties: An overview. Forsch Komplementmed 2009; 16: 79-90. https://doi.org/10.1159/000207196
  2. Serrano J, Puupponen PR, Dauer A, et al. Tannins: Current knowledge of food sources, intake, bioavailability and biological effects. Mol Nutr Food Res 2009; 53(2): S310-S329. https://doi.org/10.1002/mnfr.200900039
  3. Kitic D, Jovanovic T, Ristic M, et al. Chemical composition and antimicrobial activity of the essential oil of Calamintha nepeta (L.) Savi ssp. glandulosa (Req.) P. W. Ball from Montenegro. J Essent Oil Res 2002; 14(2): 150-152. https://doi.org/10.1080/10412905.2002.9699802
  1. Kürkçüoglu M, Iscan G, Ozek T, et al. Composition and antimicrobial activity of the essential oils of Calamintha betulifolia et Bal. J Essent Oil Res 2007; 19: 285-287.  https://doi.org/10.1080/10412905.2007.9699282
  1. Btissam R, Fatima E, Kamal E, et al. Composition and antibacterial activity of hydro-alcohol and aqueous extracts obtained from the Lamiaceae Pharmacogn J 2018; 10(1): 81-91. https://doi.org/10.5530/pj.2018.1.16
  2. Silva CB, Simionatto E, Hess SC, et al. Chemical composition and allelopathic activity of the volatile oil of Hydrocotyle bonariensis Lam (Araliaceae). Quim Nova. 2009; 32(9): 2373 -2376. https://doi.org/10.1590/S0100-40422009000900026
  3. Gobbo-Neto L, Lopes NP. Medicinal plants: Factors of influence on the content of secondary metabolites. Quim Nova 2007; 30(2): 374-381. https://doi.org/10.1590/S0100-4042200700020002